Etiologic Risk Group (see link below):Appendix C-VI. Extrachromosomal Elements of Gram Positive Organisms
*/Disease Information (sort of like the Intro to your Mini Research Write up):Listeriosis is a potentially fatal disease caused by the bacterial organism Listeria monocytogenes. It is unique from other bacterium, in that it can grow in colder temperatures. L. monocytogenes, in recent years, has been responsible for multiple small outbreaks of foodborne illnesses, many of which have resulted in fatalities. As a result, L. monoctyogenes is a growing concern to public health around the world.
*Target (protein/gene name): Thioredoxin reductase
*NCBI Gene # or RefSeq#: NC_003210.1 (NCBI)
*Protein ID (NP or XP #) or Wolbachia#: NP_466001.1
*Organism (including strain): Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
Etiologic Risk Group (see link below): Appendix C-VI. Extrachromosomal Elements of Gram Positive Organisms
*/Disease Information (sort of like the Intro to your Mini Research Write up): Listeriosis is a potentially fatal disease caused by the bacterial organism Listeria monocytogenes. It is unique from other bacterium, in that it can grow in colder temperatures. L. monocytogenes, in recent years, has been responsible for multiple small outbreaks of foodborne illnesses, many of which have resulted in fatalities. As a result, L. monoctyogenes is a growing concern to public health around the world.
Link to TDR Targets page (if present): Not Present
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/protein/16804516
Essentiality of this protein:
In S. aureus, has been shown to be essential for growth (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC305758/)
Essential for the Thioredoxin system in multiple organisms. Thioredoxin system acts as a hydrogen donor to ribonucleotide reductase, which is essential for DNA synthesis. (http://www.sciencedirect.com/science/article/pii/0076687995520236)
Essential in E. coli, along with Glutaredoxin Pathway, in keeping multiple proteins functional by reducing disulfide bonds. (http://www.jbc.org/content/272/25/15661.full)
Complex of proteins?: dimer
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html)
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
“Thioredoxin reductase: A target for gold compounds acting as potential anticancer drugs” (http://www.sciencedirect.com/science/article/pii/S0010854509000770)
“New uses for old drugs. Auranofin, a clinically established antiarthritic metallodrug, exhibits potent antimalarial effects in vitro: Mechanistic and pharmacological implications” (http://www.sciencedirect.com/science/article/pii/S0014579308001361)
*EC#: 1.8.1.9
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.8.1.9
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): 5,5'-dithiobis(2-nitrobenzoic acid) + NADPH = 2-nitro-5-thiobenzoate + NADP+ (coupled assay)
-- link to Sigma (or other company) page for assay (see Sigma links below): http://www.sigmaaldrich.com/catalog/product/sigma/t7915?lang=en®ion=US
-- -or link (or citation) to paper that contains assay information: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/thioredoxinred.pdf
-- links to assay reagents (substrates) pages.
1.) Potassium phosphate monobasic (P5379 SIGMA-ALDRICH) http://www.sigmaaldrich.com/catalog/product/sial/p5379?lang=en®ion=US
2.) Ethylenediaminetetraacetic acid disodium salt dihydrate (ED2SS SIGMA-ALDRICH) http://www.sigmaaldrich.com/catalog/product/sial/ed2ss?lang=en®ion=US
3.) β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (N6505 SIGMA) http://www.sigmaaldrich.com/catalog/product/sigma/n6505?lang=en®ion=US
4.) Bovine Serum Albumin (A4503 SIGMA) http://www.sigmaaldrich.com/catalog/product/sigma/a4503?lang=en®ion=US
5.) 0.1% (w/v) Thioredoxin Solution (Thioredoxin) http://www.sigmaaldrich.com/catalog/product/sigma/t0910?lang=en®ion=US
6.) 100 mM 5'5-Dithio-bis(2-Nitrobenzoic Acid Solution) (DTNB) http://www.sigmaaldrich.com/catalog/product/aldrich/d218200?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #
β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH)
Sigma catalog #: N6505
Quantity: 25mg
Price: 95.40
Structure (PDB or Homology model)
Homology Model: Thioredoxin reductase (trxB) from Staphylococcus aureus (64% identical amino acid sequence) http://www.rcsb.org/pdb/explore/explore.do?pdbId=4GCM
-- PDB # or closest PDB entry if using homology model: no PDB available (L. monocytogenes)
-- For Homology Model option: 4GCM (S. aureus)
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 95%
---- Max % Identities: 64%
---- % Positives 78%
---- Chain used for homology:
>4GCM:A|PDBID|CHAIN|SEQUENCE GMTEIDFDIAIIGAGPAGMTAAVYASRANLKTVMIERGIPGGQMANTEEVENFPGFEMITGPDLSTKMFEHAKKFGAVYQ YGDIKSVEDKGEYKVINFGNKELTAKAVIIATGAEYKKIGVPGEQELGGRGVSYCAVCDGAFFKNKRLFVIGGGDSAVEE GTFLTKFADKVTIVHRRDELRAQRILQDRAFKNDKIDFIWSHTLKSINEKDGKVGSVTLTSTKDGSEETHEADGVFIYIG MKPLTAPFKDLGITNDVGYIVTKDDMTTSVPGIFAAGDVRDKGLRQIVTATGDGSIAAQSAAEYIEHLNDQA
Current Inhibitors:
APR-246/PRIMA-1MET (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920950/)
Ebselen [2-phenyl-1,2 benzisoselenazol-3(2H)-one] (http://www.fasebj.org/content/27/4/1394.long)
RITA (NSC 652287) (http://www.tandfonline.com/doi/abs/10.4161/cc.8.21.9977#.VS_uvxPF-is)
Expression Information (has it been expressed in bacterial cells):Can be “indirectly” expressed in E. coli. (http://www.sciencedirect.com/science/article/pii/S0022283699930851)
Purification Method: 2′,5′-ADP-Sepharose purification (specific affinity purification for NADP(H)-binding proteins)(http://www.sciencedirect.com/science/article/pii/S0022283699930851)
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MASEEKIYDVIIIGAGPAGMTAALYTSRADLDTLMIERGVPGGQMVNTAEVENYPGFDSI LGPDLSDKMLSGAKQFGAEYAYGDIKEVVDGKEFKTVTAGSKTYKARAIIIATGAEHRKL GAAGEEELSGRGVSYCAVCDGAFFKNRELIVVGGGDSAVEEGTYLTRYADKVTIVHRRDK LRAQQILQDRAFKDEKVDFIWNSTVEEIVGDGKKVTGAKLVSTVDGSESIMPVDGVFIYV GLVPLTKAFLNLGITDDEGYIVTDEEMRTNLPGIFAAGDVRAKSLRQIVTATGDGGLAGQ NAQKYVEELKESLEAEAAK
*length of your protein in Amino Acids 319
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 34.1736 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 23505
Abs 0.1% (=1 g/l) 0.688, assuming all pairs of Cys residues form cystines
Ext. coefficient 23380
Abs 0.1% (=1 g/l) 0.684, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGGCTAGTGAAGAAAAAATTTATGATGTGATTATTATTGGGGCGGGGCCTGCTGGAATGACAGCAGCTT
TATATACTTCCCGTGCAGATTTAGATACGTTAATGATTGAACGCGGTGTACCCGGTGGACAAATGGTTAA CACAGCTGAGGTAGAAAATTACCCAGGATTTGATAGCATTTTAGGACCAGATTTGTCCGACAAGATGCTT AGTGGTGCGAAACAATTTGGAGCTGAGTATGCTTATGGTGATATTAAAGAAGTTGTTGACGGTAAAGAAT TTAAAACAGTAACAGCTGGGTCGAAAACCTACAAGGCTCGGGCAATTATTATTGCGACTGGAGCTGAACA CCGTAAACTTGGCGCGGCTGGTGAAGAAGAACTTAGTGGACGTGGTGTTTCTTACTGCGCAGTCTGTGAC GGAGCATTCTTCAAAAATCGCGAATTAATTGTGGTTGGCGGCGGAGACTCTGCAGTGGAAGAAGGAACCT ACTTGACTCGTTATGCAGATAAAGTAACCATTGTACACCGTCGTGATAAATTGCGAGCACAACAAATTTT ACAAGATCGTGCTTTTAAAGATGAAAAAGTTGATTTCATTTGGAATAGTACAGTAGAAGAAATTGTTGGC GATGGTAAAAAAGTAACAGGAGCAAAACTTGTTTCTACTGTGGATGGCTCCGAATCGATTATGCCAGTAG ATGGCGTTTTCATCTATGTTGGACTTGTTCCACTTACGAAAGCATTCTTAAATTTAGGTATTACAGATGA TGAAGGATACATCGTAACGGACGAAGAAATGCGTACAAATCTTCCTGGTATTTTTGCAGCAGGTGATGTT CGTGCGAAAAGCTTACGCCAAATTGTAACAGCTACAGGCGACGGCGGACTTGCTGGACAAAATGCTCAAA
AATATGTAGAAGAATTAAAAGAGTCGCTAGAAGCTGAGGCGGCTAAATAA
*GC% Content for gene: 41.6%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): not available
*GC% Content for gene (codon optimized): not available
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**